MODULE 5 - Putting Microbes to Work in the Environment Flashcards
why do biofilms have to be on wet surfaces?
they have to have a moisture content so the bacteria can survive
what are some nutrients and environmental factors that form gradients?
water
pH
temp.
oxygen
pressure
radiation
how can a bacteria survive starvation through morphological changes?
endospores (metabolically dormant, resistant to heat)
nucleoid condensation (nucleoid associated proteins bend DNA so it can condense, stress the bacteria and they switch to starvation mode where gene transcription changed, so stress response pathways activated and growth slowed)
how can bacteria survive starvation with starvation proteins?
transpeptidases which cause peptidoglycan cross-linking and thickening of cell wall
chaperones which prevent denaturation and help renature damaged proteins
why are starved cells hard to kill?
the can survive for years
can become more virulent
how does formation of persister cells occur?
small subset of cells which are spontaneously dormant (non-growing) even with nutrients available
starvation triggers persister cells through nutrient depletion leading to stress adaptation
formation of persister cell occurs in exponential phase
why are persister cells important?
they can’t be treated with antibiotics
they don’t harbour antibiotic ressitance genes and instead express antibiotic tolerance phenotype
they are tolerant to the immune system and so are all together hard to eradicate
what is the difference between antibiotic resistance and antibiotic tolerance?
resistance based on genetic mutation on chromosome, while tolerance means the bacteria overcomes the antibiotic but isn’t resistant to it
what’s worse than having persister cells infecting you?
having persister cells in a biofilm infecting you
biofilm formation can also enhance persister cell formation by stressing bacteria
what are the stages of biofilm development?
reversible attachment
irreversible attachment (EPS starts to be secreted)
maturation
maturation and dispersion
where in the biofilm are you likely to find bacteria with a tolerant phenotype?
in the deeper levels where they are under more stress (O2 and nutrient limitations)
when bacteria don’t grow that well they might randomly start mutating chromosome, if antibiotic treatment happening they especially can develop R genes
can biofilms have multiple type of microorganism in them?
yes
they can be individual or polymicrobial (e.g. bacteria, fungi)
in what stage of biofilm formation are bacteria most vulnerable to eradication?
during reversible attachment
during irreversible attachment bacteria much more tolerant to antibiotics, immune system and starvation and generally cannot be eradicated by antibiotics alone
what is extracellular polymeric substances and what does it do?
major component of a biofilm (50-95% of dry weight)
chemical composition may vary between different strains/enviros but is primarily polysaccharide
protects from desiccation, antibiotics, toxins, immune cells
maintains integrity of biofilm and binds essential nutrients (making local rich environment)
what are the advantages of biofilms?
physical attachment (in moving enviros like river, GI tract, bloodstream)
this beneficial as allows it to stay in one place, substrate may provide nutrients, extracellular enzymes that solubilise don’t get diluted quickly, nutrients may be higher in biofilm than enviro
what is the medical significance of biofilms?
delay wound healing
increase risk of infection (chronic, polymicrobial)
protects from body’s immune response (inflam response may induce biofilm formation)
providing nutrients in form of exudate (from dead immune cells)
damages healing tissue
how common are biofilm bacteria involved in chronic (non-healing) wounds?
biofilm bacteria in 60-90% chronic wounds
these wounds are stuck in inflammatory phase of healing and cannot progress further
often not easily realised and bacteria feed on host response
how can host immune response worse biofilm formation in wound?
neutrophils and macrophages come to wound to release proteases (e.g. matrix metalloproteinases (MMPs)) as these degrade dead tissue and extracellular matrix proteins
problem is when too many pathogens in wound more and more neutrophils come and produce more metalloproteases which provides more nutrients for bacteria by degrading dead tissue
in normal physiological conditions MMP levels controlled
what are matrix metalloproteinases (MMPs)?
proteases which require metal as co-factor for catalytic activity to occur
factor in abnormal healing in chronic wounds due to over production and increased protease activity
unbalanced activity facilitates colonisation and proliferation in chronic wounds
bacteria also secrete them so in bad wound lots of tissue being degraded thus lots of nutrients for bacteria
how are biofilms a problem for our teeth?
dental plaque is a biofilm and formation damages tooth and causes receding gums and bad breath
causes periodontal disease, dental caries
biofilms on teeth a symptom
flossing/brushing regularly prevents this
what are some biofilm infections that form from objects?
catheter insertion causing UTIs, vascular disease (bloodstream)
catheter contaminated often through caregivers hand
piercings, tattoos and brandings
how can we prevent biofilm formation on wounds?
regularly clean and debride wound tissue, remove biofilm material, necrotic tissue, foreign material in order to prevent biofilm maturation
sometimes difficult as biofilms might be attached to healthy tissue
most effective in early stages
how can we manage biofilm once it has formed in a wound?
increase frequency of debridement
cannot completely remove biofilm
once removed prevent re-establishment, otherwise this can happen within 24h
how do we treat bacterial biofilms once they are formed in wounds?
biofilms become resistant to most antimicrobials within 48-96hrs
clinical management requires complete removal of infected area
if not possible, attacking on regular schedule may force biofilm detachment, make bacteria susceptible for treatment and host defences HOWEVER can cause systemic infection e.g. sepsis, bacteraemia which are more virulent infections
what are the stages of biofilm development?
initial attachment phase
primary colonisation phase
climax community phase
what influences adhesion of the first few bacteria in a biofilm?
linked to environmental cues and quorum sensing
they also produce biosurfactants to break down water content in an environment
what are the surfaces bacteria adhere to before forming biofilm?
attachment often occurs on rough surfaces e.g. a crack to get stuck in
if on smooth surface could be due to flow boundary layer where flow pushes bacteria against surfaces
describe the initial attachment phase?
microbes attach to a surface or themselves
attachment helped with flagella, filaments (help bacteria recognise surface structures), fimbriae (little charged hairs which are attracted to certain surfaces) and pili
mobile bacteria in aqueous phase become loosely attached via electrostatic or hydrophobic interaction w surface
describe the primary colonisation phase?
permanent attachment and symbiotic community
production of EPS and extracellular DNA which is the foundation of the biofilm
interaction with substrate which is mediated by production of extracellular polymers by colonising bacteria
once attached to surface active growth begins, micrcolonies form on substrate, further growth continues to cover entire surface
describe the climax community phase of biofilm development?
develops within seven days, growth continues until steady state
once mature biofilm may contain bacteria that can’t even attach to substrate or survive initial nutritional restrictions but other bacteria clutched up (polymicrobial)
what is biofouling?
accumulation (usually a biofilm) of bacteria on a surface they are not wanted on e.g. industrial equipment, ship
what is economies of scale?
ideally you spend lots on equipment at first and then over time produce more product and so it pays off the investment
so you have to find sweet spot where investment pays off as equipment lasts longer
what are common places in industry where biofilm formation is a problem?
power plants, air conditioning, food processing, oil refining
biofilms cause major production disruptions
what are some industrial examples of financial losses due to biofilms?
food industries lose large production due to contamination
fouling of pipes leads to friction and plant failure through corrosion and lesser flow
heat exchanger surfaces lead to inefficient heat exchange
cleaning requires closing of entire plant and scrubbing of plumbing which decreases production
how are biofilms a major problem for the dairy industry?
milk highly perishable as has high nutrient content so easy for microbes to grow
sterile in udder cells
biofilms form at air-liquid interface and are usually bacillus species (floating biofilms)
outline the bacillus genus and how it is bad for dairy industry?
gram-positive rod shaped
highly detrimental as cause dairy spoilage and illness
associated with animal udders and then spread through diary production systems
prod heat resistant endospores allowing persistence through pasteurisation and then biofilm formation
what are some biofilm components produced by bacillus?
exopolysaccharides
amyloid-like fibres (non-soluble proteins) - soluble proteins which fold into insoluble fibres allowing further resistance
what are some common methods of cleaning equipment for biofilm control and what are some problems with this?
use cheap chemical agents such as chlorine, NaOH, acid
chlorination is most common methods however problem is some microbes resistant and chlorine also not good enough to eliminate biofilms as leaves EPS matrix intact
what are some examples of damage caused to at home equipment from biofilms?
rubber seals in washing machine and dishwasher
what is the dairy plant cleaning in place (CIP) procedure?
cold water and acid wash immediately after milking
hot water and NaOH wash to remove adhered residues at least twice a week
cold water acid wash after each alkali wash to remove minerals, kill remaining bacteria and neutralise alkali solution
final wash w detergent
how acids destroy cells?
denature proteins, extremely reactive and break apart chemical structures, upon exposure cells die but don’t disintegrate
how do alkalis destroy cells?
accept protons (i.e. rip them the fuck off other molecules), found in cleaners and bleach
burns from alkalis way worse than acid burns as they liquify biological material
how does biofouling cause problems in NZ involving marine pests and disease?
underside of bots prone to bacteria attachment and biofilm formation
this can spread harmful bacteria
to prevent this must clean hull of ship, also can use antifouling coating
vessels must show evidence of biofouling management
outline some key forms of biofouling management?
clean niche areas e.g. protrusions, recesses in deck, unpainted parts of hull
maintenance procedures e.g. paint w antifouling paints, marine growth prevention systems (e.g. approved chemicals), steam blow-out pipes
how are certain surfaces anti-fouling?
reagents/dispersants such as repelling agents, slippery surface coatings and nanoscale surface topologies
how can biocides prevent fouling on the hulls of boats?
chemical removal of biofouling and can be incorporates into antifouling coatings
specifically kills bacteria, fungi and algae
e.g. tributyltin (TBT)
what is tributyltin (TBT)?
a common biocide which is now banned
component of paint and slowly released into environment
very poisonous to marine life e.g. shellfish
highest concentrations in harbours where boats with this coating moored
do antifouling coatings kill microorganisms?
nah they just prevent them from attachment
what are the three main classes of antifouling coatings?
hydrophobic coating
hydrophilic coating
polymer coating
what are hydrophobic coatings?
repel water
low friction and surface energy
smooth surface
problem is no mechanical strength and only short term stability (so have to be reapplied frequently)
e.g. silicone or umbrellas/rain jackets
what are hydrophilic coatings?
attract water
highly hydrated zwitterions composed of glycine betaine get saturated w water molecules and repel organisms, enzymes and EPS
low friction and work better then hydrophobic surfaces in regards to biofilm formation
however not commercially available
what are polymer coatings?
poly(ethylene glycol) - PEG
hydrophilic but water soluble so difficult to use as a coating in aqueous enviro
research on mussel adhesive proteins (MAPs) prod by blue mussel and adding this to PEG to make it more adhesive
what are some antifouling examples from nature?
bacteria don’t attach to shark skin, butterfly wings, seaweed
so there are antimicrobial antifouling compounds in nature we haven’t discovered which could be mimicked for engineering purposes
what are some coatings we could add to our teeth to prevent oral biofilms?
Microparticles w quorum sensing inhibitors to block biofilm formation
calcium-binding polymer coated microparticles
could allow sustained release of QS inhibitors
how did oil get dispersed far as fuck when deepwater horizon happened?
grease-cutting soaps such as dispersants break oil into small bits and so it merged with deep sea water and sunk
it also spread as dragged down by marine snow and wind/currents pushed it far away
when they couldn’t find like a quarter of the oil spilt from deepwater horizon, what happened to it?
hydrocarbon-degrading bacteria in the sediments mineralised it to CO2 or immobilised it as biomass
what is bioremediation?
engineered process using microbes to break down environmental pollutants from a spillage
microbes involved are often extremophiles as they can tolerate high levels of organic solvents
what are hydrocarbonoclastic bacteria?
two main species: Alcanivorax borkumensis
Oleispira antarctica
can break down hydrocarbons to CO2 and energy however cannot break down polyaromatic hydrocarbons as complex structure
require specific O2, nitrogen, phosphorus and temp to live and often this not correct in oil spill zones of ocean
describe Alcanivorax borkumensis?
aerobic hydrcarbonoclastic bacterium which propagates in seawater containing crude oil
oil leakage increases phosphorous and nitrogen which is natural nutrient for bacteria = better growth
produces biosurfactants which break down water surface tension so oil forms droplet
bacterial then covers droplets w biofilm
describe Oleispira antarctica?
aerobic/anaerobic hydrocarbonoclastic bacterium which lives in cold marine water
can degrade oil in cold and deep water
growth temp. between 1-15 degrees celsius
what are some examples of fungi we could use for bioremediation?
oyster mushroom - eats diesel and other petroleum products producing CO2 and H2O as result
white rot fungus - can break down polyaromatic hydrocarbons that the bacteria couldnt prod H2O2
what are some of the drawbacks of using fungi for bioremediation?
they grow slow af so slow process
they will also degrade biological material so other environmental consequences (this one applies to bacteria too)
what are the two types of bioremediation?
biostimulation - environmental modification by adding nutrients to stimulate certain microbes or aerating soil/water
bioaugmentation - addition of microbes
what are the advantages of biostimulation?
cheaper
natural process so publicly acceptable
less disturbing to enviro
destroys wide range of contaminants
what are the disadvantages of biostimulation?
often unpredictable or unsuccessful
physical environment challenging due to pH, temp, O2 etc. (so adding nutrients to certain enviro may not help growth as other factors limiting)
concentration dependent
slow process
biological competition
what are some problems with bioaugmentation?
bacteria require certain nutrients that might not be present
not enough bacterial numbers and they just get diluted in enviro
could disturb ecological niches
what is bioaugmentation good for?
degrading specific soil or groundwater contaminants such as chlorinated solvents or petroleum
describe the Pseudomonas species?
rod-shaped bacterial species
opportunistic pathogen
some can protect crops from pests and disease
some can cause precipitation by cooling vapour and some can utilise petroleum products
so can be used to clean up spills
what is biodegradation?
natural break down of everything which replenishes ecosystem
microbes degrade organic matter slowly
what are the stages of biodegradation?
- biodeteriation - surface level degradation where material properties modified
- biofragmentation - degrades polymers to oligomers and monomers; if anaerobic prod methane, if aerobic prod CO2 and H2O
- assimilation/mineralisation - microbes use the products of prev step
what are some common plastic degrading microbes?
Penicillum, Aspergillus, Pseudomonas sp.
these can degrade non-biodegradable plastics and consume for growth by secreting PETase enzymes
slow degradation time so no commercial scale
also some gut microbes and also mealworms
what are xenobiotics?
foreign compounds introduced into environment by humans
give an example of a xenobiotic and how it is problematic?
DDT is a pesticide which kills insects and thus can control malaria and typhus outbreaks
still present in soil, has detrimental reproductive impacts (oestrogen blocker) and causes cancer
why are xenobiotics usually so hard to get rid of?
they usually stable for a long time
have complex ring structures and stable C-halogen bonds which are highly chlorinated
very recalcitrant to microbial attack
shortage of enzymes to get rid of them
how can we degrade DDT?
reduce the organochlorines (used in pesticides)
DDT converted to DDE and then to DDD
this can be done with sulcate-reducing bacterium such as Clostridium sp.
what is PCB?
a very chlorinated and very recalcitrant xenobiotic used in coolant fluids
it may not degrade at all
takes a long time to test for biodegradation
what happens to waste material from an organism in the environment?
either used directly by another organism or converted
what are common liquid wastes humans produce?
shower water
urine
washing-machine water
trade wastes and other industrial wastes
what are some solid wastes humans produce?
human faeces
kitchen waste
(nowhere near as much liquid waste and gets mixed w water for transport e.g. flush toilet)
what is mixed domestic sewage?
our combined liquid and solid wastes
what does waste water include and what are there in high levels?
includes sewage, industrial and agricultural effluent, street runoff collected in drains
contains high levels of organic matter, heavy metals, nutrients and microorganisms
what are some common diseases that are spread in wastewater?
typhoid
cholera
COVID-19 (not really common but relevant)
what are the main microbes of human origin in wastewater?
coliforms such as E. coli
presence of E. coli indicates faecal matter from warm-blooded animals (hence use as indicator bacteria)
some disease causing pathogens e.g. Salmonella can spread and re-enter human food chain e.g. through shellfish
can contaminate groundwater
what are the goals of wastewater treatment?
eradicate any human pathogen
seperate wastewater into sludge and a dissolved fraction containing water, organic material, bacteria and salts
reduce organic load (microbes, insoluble debri, soluble organic matter)
remove chemical compounds e.g. N and P
make it safe for marine disposal
what occurs in primary treatment of wastewater?
wastewater enters treatment plant
passed through milliscreens which sieve for large solids (anything that doesn’t go through goes to landfill)
then through grit chambers which slow the flow and into primary clarifier
what does the primary clarifier do?
separates suspended solids from liquid
gravity pulls smaller particles into sludge and lighter solids float at top and accumulate as scum
sludge goes into waste disposal area
what occurs in secondary treatment of wastewater?
aeration basins blow air in to keep mixture in motion and provides oxygen
environmentally friendly bacteria (large consortia) feed on nutrients in wastewater e.g. fats, sugar. ammonia
bacterial clusters form as they break down waste; can form biofilms which are removed via settlement
secondary clarifier separates bacterial clusters from liquid through flocculation
activated sludge pumped back into aeration basin to provide more bacteria for breakdown
separation of solids from liquids and then centrifugation (liquid back into aeration basin and sludge disposed)
what occurs in final/tertiary treatment?
disinfection to kill any remaining microorganisms; traditionally used chlorine (effluent <0.5mg/L Cl)
filtration (e.g. gravel or sand)
UV light disinfection
release into harbour
how can we find microbes for remediation?
isolation and characterisation of microbes from unique habitats
traditional lab conditions that test microbial consortia to perform bioremediation
culture-based techniques (problematic cause so many microbes unculturable)
DNA-based techniques are what we now turning towards
what are some DNA-based techniques for identification of microorganisms?
16S rRNA sequencing
Omics-approaches
what are the omics-approaches?
discover novel microbes not accessible w traditional culturing
explore metagenomes of contaminated environmental samples for their microbial communities (gives insight on diversity)
allows to design and develop efficient strains of microbes e.g. for better metabolism of xenobiotics
what is Pseudomonas putida?
soil bacterium and plant coloniser
adapted lifestyle to harsh environmental conditions and stresses
remarkable metabolic and physiological robustness
uses wide variety of C and N sources
can use omics to engineer new optimised strains
what is metabolic engineering?
purposeful modification of cellular networks including metabolic, gene regulatory, and signalling networks to achieve desirable goals e.g. enhanced production of metabolites, increase intracellular levels of essential precursors, nutrient source uptake and by-product formation
how can metabolically engineer Pseudomonas putida for better bioremediation?
synthetic biology tools combined with omics data can be used to re-purpose the central carbon metabolism leading to synthesis of new chemical structures
what are some problems with bioengineering and how can we overcome them?
production of dead-end substrates e.g. reactive intermediates which cannot be metabolised so bacteria stop growing
overcome by restructuring existing pathways and developing new ones with different enzymes
what are some problems with bioengineering and how can we overcome them?
production of dead-end substrates e.g. reactive intermediates which cannot be metabolised so bacteria stop growing
overcome by restructuring existing pathways and developing new ones with different enzymes
what are DCA and TCP examples of?
carcinogenic and mutagenic xenobiotics which cause reproductive effects
highly recalcitrant in enviro and initially used as degreasing agent
spread via groundwater flows
how can bacterial degradation of DCA occur and how could we use this for large scale industrial applications?
Xanthobacteri autotrophicus pathway can degrade it by:
haloalkane dehalogenase hydrolyses one C-Cl bond
dehydrogenase produces chloroacetic acid
dehalogenase converts this to glycolic acid which then can turn to CO2
if we introduce this pathway into P. putida could be used for large scale applications
how can bacterial degradation of TCP occur?
natural organisms cannot mineralise TCP, however similarly structured compounds are biodegradable
protein and metabolic engineering to construct microbes with improved catabolic activities
how could we engineer DCP degrading enzymes to degrade TCP?
haloalkane dehalogenase (DhIA) catalyses first step in DCA degradation and could be engineered to have wider substrate range
this engineering could be done through site-directed mutagenesis to improve activity or modification of tunnel proteins
very strict here but some techniques no approved e.g. more protein-protein expression which is way more safe and cost effective than other methods
